Microprocessor Activity Controls Differential miRNA Biogenesis In Vivo

Abstract

In miRNA biogenesis, pri-miRNA transcripts are converted into pre-miRNA hairpins. The in vivo properties of this process remain enigmatic. Here, we determine in vivo transcriptome-wide pri-miRNA processing using next-generation sequencing of chromatin-associated pri-miRNAs. We identify a distinctive Microprocessor signature in the transcriptome profile from which efficiency of the endogenous processing event can be accurately quantified. This analysis reveals differential susceptibility to Microprocessor cleavage as a key regulatory step in miRNA biogenesis. Processing is highly variable among pri-miRNAs and a better predictor of miRNA abundance than primary transcription itself. Processing is also largely stable across three cell lines, suggesting a major contribution of sequence determinants. On the basis of differential processing efficiencies, we define functionality for short sequence features adjacent to the pre-miRNA hairpin. In conclusion, we identify Microprocessor as the main hub for diversified miRNA output and suggest a role for uncoupling miRNA biogenesis from host gene expression.

Microprocessor dynamics shows co- and post-transcriptional processing of pri-miRNAs

“…1B;Conrad et al 2014). While this approach provides insight into the average processing efficiency of steady-state primiRNA associated to chromatin, it cannot address the dynamics of this process.…”

“…We assessed whether there is a correlation between the expression level of pri-miRNAs and the processing kinetics (Supplemental Fig. 1) based on chromatin-associated RNA-sequencing data from Conrad et al (2014). The median expression level for the three groups are comparable, albeit higher than the average expression of pri-miRNAs in HEK293 cells, reflecting that the sequencing-based assay captures the highly expressed pri-miRNAs with highest confidence.…”

“…These processing efficiencies can be recapitulated using quantitative PCR of individual pri- miRNAs. Using primers spanning the processing site we can determine the relative amounts of unprocessed pri-miRNAs, and taking the ratio to the level of total pri-miRNA (processed + unprocessed) we can estimate the processing efficiency for single pri-miRNA transcripts, as described in Conrad et al (2014). Representative results from the PCR quantification of pri-miRNA processing for miR-221 and let-7a-1 are shown in Figure 1D.…”

“…Several features, such as sequence motifs around the precursor miRNA (pre-miRNA) hairpin in the primary miRNA (pri-miRNA) transcript (Auyeung et al 2013;Conrad et al 2014) and within the hairpin loop (Auyeung et al 2013), have been shown to be involved in processing efficiency in mammals. Furthermore, early studies of primiRNA transcripts have reported a cotranscriptional processing by the Microprocessor complex (Kim and Kim 2007;Morlando et al 2008), using mostly in vitro assays and studies of single endogenous examples.…”

“…We have previously shown that sequencing of the chromatin-associated RNA can reveal the steady-state processing efficiency of individual pri-miRNAs within the cell, demonstrating pri-miRNA processing as one of the most important factors for determining the level of mature miRNAs (Conrad et al 2014). To further follow processing of pri-miRNAs endogenously over time without constraints of their cellular localization or differential transcription, we pulse-labeled RNA with bromouridine (BrU) and followed its processing during a 1-h chase (Paulsen et al 2013).…”